The role of fibromodulin in myocardial fibrosis in a diabetic cardiomyopathy rat model

Figures

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  Fig. 1. Fibrosis indicators and Fmod increased in DCM rat hearts and RPCFs treated with high glucose. (A) HE staining results. (B) Masson staining stained the collagen fiber blue, the muscle fiber cytoplasm red and the nucleus brown. Note the irregular arrangement of myocardial fibers and more blue staining in the DCM group. (C1, C2) Immunohistochemistry analysis. Brown indicates positive expression of fibrosis indicators. The vertical coordinate represents the MOD value in image pro plus, version 6.0, where higher values represent stronger positive expression. (D, E) RT‐qPCR results of rat hearts and RPCFs, respectively, and the results represent relative mRNA expression levels, which were normalized to the reference gene (Tubb5). (F1, F2; G1, G2) Western blot results of rat hearts and RPCFs, respectively, and the results represent relative protein expression levels, which were normalized to the reference gene (β‐tubulin). (A, left; B, left) Magnification 200×; scale bar = 100 μm. (A, right; B, right) Magnification 400×; scale bar = 50 μm. (C1) Magnification 200×; scale bar = 100 μm. The data are presented as the mean ± SD or median (Q1, Q3). An unpaired t test was used with respect to…

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  Fig. 2. Col1a1, α‐SMA and Col1a1/Col3a1 ratio increased and Col3a1 decreased in ov‐Fmod RCFs, whereas the opposite trend was found for the si‐Fmod RCFs RT‐qPCR (A, C) Col1a1, Col1a1/ Col3a1 and Acta2 increased in the ov‐Fmod group and decreased in the si‐Fmod group, whereas Col3a1 decreased in the ov‐Fmod group and decreased in the si‐Fmod group. Similarly, western blot results (B1, B2, D1 and D2) showed that Col1a1, Col1a1/ Col3a1 and α‐SMA increased in the ov‐Fmod group and decreased in the si‐Fmod group, whereas Col3a1 decreased in the ov‐Fmod group and decreased in the si‐Fmod group. The data are presented as the mean ± SD except max/min in (A) (right). Welch's t test was used with respect to all indicators in (A) (left), Fmod in (B2), Fmod, Col1a1, Col3a1 and Col1a1/Col3a1 in (C), and COL3A1 and α‐SMA in (D2). A Mann–Whitney test was used in (A) (right). An unpaired t test was used with respect to COL1A1 and COL1A1/COL3A1 in (B2), Acta2 in (C), and FMOD, COL1A1 and COL1A1/COL3A1 in (D2). Compared with Control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, N = 3.

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  Fig. 3. Changes in transcriptome of fibrosis‐related differential genes of Fmod transfected cells six samples of two groups were used in transcriptome analysis (n = 3, each group). (A) Boxplot of FPKM values of genes across samples showing consistent levels of gene expression between groups of samples. Boxplots showing five‐number summaries (minimum, first quartile, median, third quartile, maximum). (B) The clustered heatmap shows the DEGs. The red in the graph indicates relatively highly expressed protein‐coding genes and the blue indicates relatively lowly expressed protein‐coding genes. (C) The volcano plot shows the overall distribution of genes in the two groups. Red and blue are the significantly different genes. (D) GO chord plots were constructed to highlight core targets related to fibrosis. (E) Bar graph demonstrating the top30 entries of GO enrichment analysis of DEGs. (F) Bubble plot showing the top20 entries of KEGG enrichment analysis. Col15a1 was marked with red box in (D), (E) or (F).

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  Fig. 4. Change of Col15a1 in DCM rat model and in vitro experiment and correlation of plasma FMOD and COL15A1 levels in DCM rats. RT‐qPCR results (A) and ELISA results (B) showing that Col15a1 was decreased in the DCM animal model, high glucose‐treated RPCF, and overexpressing‐FMOD RCF. Col15a1 rose in si‐Fmod RCF. ELISA results (C) showing that plasma FMOD levels increased in DCM rats. Correlation analysis results (D) showing a negative correlation between plasma FMOD levels and COL15A1 levels in the diabetic rat model. The data are presented as the mean ± SD. Welch's t test was used with respect to DCM in (A), ov‐Fmod and siFmod in (B), and DCM in (C). An unpaired t test was used with respect to ov‐Fmod and si‐Fmod in (A) and DCM in (B). Brown‐Forsythe and Welch ANOVA tests were used with respect to NG, HG and OC in (A). Ordinary one‐way ANOVA was used with respect to NG, HG and OC in (B). Pearson correlation coefficients were used in (D). Compared with the control group: **P < 0.01, ***P < 0.001, ****P < 0.0001, N = 4 (animal experiment), N = 3 (cell experiment).